Purification by affinity chromatography and immunological characterization of a 110kDa component of the chick oviduct progesterone receptor

Renoir, Jack‐Michel; Mešter, Ján; Buchou, T.; Catelli, Maria-Grazia; Tuohimaa, Pentti; Binart, Nadine; Joab, Irène; Radanyi, Christine; Baulieu, Étienne-Émile

doi:10.1042/bj2170685

Cited by 68 publications

(34 citation statements)

References 22 publications

(17 reference statements)

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“…The so-called B subunit (7) of the chicken oviduct progesterone receptor was purified by affinity chromatography (8). After elution from the affinity column by [3H]R 5020 and subsequent DEAE-Sephacel chromatography, the pooled radioactive peak eluting at 0.3 M KC1 during the linear salt gradient was further concentrated by another DEAESephacel chromatography, in which it was eluted by 0.5 M KCl.…”

Section: Methodsmentioning

confidence: 99%

Receptors for glucocorticosteroid and progesterone recognize distinct features of a DNA regulatory element.

Ahe¹,

Renoir²,

Buchou³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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The chicken lysozyme gene can be induced in oviduct cells by four classes of steroid hormones, including glucocorticosteroids and progestins. The glucocorticosteroid receptor of rat liver and the progesterone receptor of rabbit uterus both bind, although with different relative affinities, to two sites in the promoter region of the chicken lysozyme gene located, respectively, between 50 and 80 and between 160 and 200 base pairs upstream of the transcription start point. Now we show that the purified progesterone binding unit of the chicken oviduct progesterone receptor (Mr 110,000, or socalled B subunit) generates a DNase I protection pattern ("footprint") in the promoter-distal site that is longer than the footprint generated by the glucocorticosteroid receptor. Methylation protection studies within the promoter-distal binding site identify four contact points for the chicken progesterone receptor and three contact points for the glucocorticosteroid receptor, of which only one is shared by both receptors. Computer graphics models allow one to envisage a different interaction of each receptor with the B form of the DNA double helix.Gene regulation by steroid hormones is mediated by an interaction of the hormone receptor with DNA elements around the regulated promoters. Such elements have been identified at variable distances from the promoter in the genes for mouse mammary tumor virus, human metallothionein IIA, chicken lysozyme, rabbit uteroglobin, and human growth hormone (for a review see ref. 1). The expression of some of these genes, as for instance the chicken lysozyme gene, can be regulated by several steroid hormones, including glucocorticosteroids and progesterone (2, 3). The question, thus, arises of whether the action of individual hormones is mediated by the same or by separate DNA regulatory elements.In the promoter region of the chicken lysozyme gene two receptor binding sites have been found, of which the promoter-proximal one (located around position -60) exhibits high affinity for the glucocorticosteroid receptor and low affinity for the progesterone receptor, whereas the promoterdistal site (around position -180) has low affinity for the glucocorticosteroid and high affinity for the progesterone receptor (4,5). No functional data are available concerning the promoter-proximal binding site, but deletions destroying the promoter-distal site result in parallel loss of inducibility by both glucocorticosteroid and progesterone, suggesting that binding of the receptor to this site is relevant for induction by both hormones (4).In exonuclease III protection experiments with the promoter-distal site we found that the region covered by the glucocorticosteroid receptor is shorter than the region protected by the rabbit uterus progesterone receptor (5), indicating that subtle differences may exist between the interaction of each receptor with the regulatory sequences. To analyze this question in more detail we decided to use the homologous progesterone receptor purified from chicken oviduct in DNa...

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Section: Methodsmentioning

confidence: 99%

Receptors for glucocorticosteroid and progesterone recognize distinct features of a DNA regulatory element.

Ahe¹,

Renoir²,

Buchou³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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“…3B). An identical control was performed in the presence of 0.8 ,g of purified B (110-kDa) subunit (17), leading to a decrease of -60% ofthe 110-kDa band (Fig. 3C).…”

mentioning

confidence: 99%

Tightly bound nuclear progesterone receptor is not phosphorylated in primary chick oviduct cultures.

Garcı́a¹,

Jung-Testas²,

Baulieu³

1986

Proc. Natl. Acad. Sci. U.S.A.

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Oviduct cells from estradiol-treated chicks were grown in primary culture. After 3-5 days of culture in medium containing estradiol, 90% of the cellular progesterone binding sites were detected in the cytosol. After exposure to [3H]progesterone at 37TC, 80% of the progesterone binding sites were found in nuclear fractions. Progesterone receptor phosphorylation was assessed after incubating the cells with [32P]orthophosphate. Receptor components were immunoprecipitated with a specific polyclonal antibody (IgG-G3) and analyzed by NaDodSO4/PAGE and autoradiography. In the cytosol, constant amounts of 32P-labeled 110-kDa subunit (the B subunit, one of the progesterone-binding components of the receptor) and of the non-steroid-binding heat shock protein hsp90 were found, whether cells had been exposed to progesterone or not. No 32P-labeled 79-kDa subunit (the A subunit, another progesterone-binding subunit) was detected. Various procedures were used to solubilize nuclear progesterone receptor (0.5 M KCI, micrococcal nuclease, NaDodSO4), and in no case was 32P-labeled B subunit detected in the extracts. However, nonradioactive B subunit was detected by immunoblot in a nuclear KCI extract of progesterone-treated cells. These results suggest that the fraction of the B subunit that becomes strongly attached to nuclear structures is not phosphorylated upon exposure of cells to progesterone.Two progesterone-binding proteins, called B (110 kDa) and A (79 kDa) subunits, have been described as components of the progesterone receptor in the chicken oviduct (1). The nontransformed form of the progesterone receptor also contains a non-progesterone-binding 90-kDa heat shock protein, hsp9o (2)(3)(4). These three components of the nontransformed progesterone receptor have been shown to be phosphorylated in vivo (2, 5). Purified A and B subunits can be phosphorylated in vitro by a cAMP-dependent protein kinase (6), and the B subunit, by the epidermal growth factor receptor tyrosine kinase (7). Purified B subunit and hsp90 can be phosphorylated by an endogenous protein kinase in highly purified progesterone receptor preparations (8).This paper reports the phosphorylation of progesterone receptor after addition of radioactive phosphate to primary oviduct cell cultures obtained from estradiol-treated chicks. Such cells multiply and respond to estradiol and progesterone in vitro (9). We studied 32P-labeling of progesterone receptor components in the cytosol and nuclear fractions from cells treated or not treated with progesterone. The tightly bound nuclear B (110-kDa) subunit, detected by radioactive hormone binding and immunoblot analysis, was not 32P-labeled, unlike the form found in the cytosol, suggesting that the hormone-dependent nuclear binding of the receptor does not depend on its phosphorylation. Labeling of Intact Cells with [3H]Progesterone. Cells (5-7 X 107) were harvested by scraping culture dishes and resuspended in 2 ml of phosphate-free phosphorylation buffer (0.9% NaCl/glucose (1 mg/ml)/1.3 mM MgCl2/2.8 mM CaCl...

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“…In 1984, Baulieu et al purified progesterone-receptor proteins from chicken oviducts pretreated with oestrogen, and studied the appearance of messenger-RNA for avidin after activation of the gene by the progesterone receptor-hormone complex (7,22,27). At the same time, they found that progesterone-receptor proteins were localized in both the tubular gland cells and the luminal epithelial cells of oestrogenpretreated chick oviducts, but were localized in the progenitor cells of immature untreated chickens, as revealed by an indirect PAP-immunohistochemistry method (4).…”

Section: Discussionmentioning

confidence: 99%

Anatomical distribution of biotin-binding protein/avidin in the hen oviduct.

Kami

Yasuda

1988

Acta Histochem. Cytochem

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Purification by affinity chromatography and immunological characterization of a 110kDa component of the chick oviduct progesterone receptor

Cited by 68 publications

References 22 publications

Receptors for glucocorticosteroid and progesterone recognize distinct features of a DNA regulatory element.

Receptors for glucocorticosteroid and progesterone recognize distinct features of a DNA regulatory element.

Tightly bound nuclear progesterone receptor is not phosphorylated in primary chick oviduct cultures.

Anatomical distribution of biotin-binding protein/avidin in the hen oviduct.

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